Abstract

Lack of effective therapy is a major problem in the treatment of pancreatic cancer. In the present study, we investigated a natural product, the extract of Pao Pereira (Pao), for its anti-pancreatic cancer effect in vitro and in vivo, either alone or in combination with the first-line chemotherapeutic drug gemcitabine (Gem). Pao induced dose-dependent apoptosis to all five tested pancreatic cancer cell lines. The combination of Pao and Gem had a synergistic effect in the inhibition of cell growth, with combination indices (CIs) <1 by Chou-Talalay's median effect analysis based on the isobologram principle. Adding Pao to Gem treatment reduced the concentration of Gem to produce an equitoxic effect on pancreatic cancer cells. In an orthotopic pancreatic xenograft mouse model, mice bearing PACN-1 tumors were treated with Pao and Gem, either alone or in combination. The progression of tumors was monitored longitudinally by imaging of live animals. While Gem did not provide significant inhibition, Pao treatment significantly suppressed tumor growth by 70-72%. Combined Pao and Gem treatment further enhanced the tumor inhibitory effect compared to Gem alone, and markedly reduced metastatic lesions in the peritoneum. Collectively, these data suggest that the extract of Pao possesses anti-pancreatic cancer activity and can enhance the effects of Gem in vitro and in vivo.

Introduction

Pancreatic cancer is one of the most lethal types of
cancer worldwide and is the 4th leading cause of cancer-related
mortality in the USA despite being responsible for only 2% of all
new cancers diagnosed (1–3). Treatment effect and prognosis for
pancreatic cancer remain dismal. Patients with this disease have an
overall 5-year survival rate of only 3–5%, which has remained
essentially unchanged over the past 30 years (1–3). This
is likely due to the limited treatment options, in addition to the
aggressive nature of this disease, and the lack of early diagnostic
tools. While surgical resection is the only potentially curative
treatment for patients with pancreatic cancer, only 15–20% of
patients have resectable disease at the time of diagnosis.
Furthermore, patients who undergo surgery (Whipple procedure) have
a perioperative mortality of 4–18% and an additional risk of post
operational complications (4–6).
Nearly 100% of patients with pancreatic cancer develop metastases
and succumb to the disease due to the debilitating metabolic
effects of unrestrained tumor growth (6). Although there have been numerous
attempts to develop improved systemic therapies of pancreatic
cancer, gemcitabine (Gem) as a single agent remains the current
standard of care (7). Gem as
first-line therapy has a 12-month survival advantage compared with
fluorouracil therapy (8). A new
Gem-free regimen FOLFIRINOX combining 5-fluorouracil, leucovorin,
irinotecan and oxaliplatin provided a 5-month survival benefit over
Gem (9). However, this regimen
added adverse effects. Lack of effective therapeutic options, lack
of adjuvant therapy, significant side-effects with existing
chemotherapies and radiation therapies or their combinations remain
major problems in the treatment of pancreatic cancer.

Natural products have long been proven a
considerable resource for bioactive anticancer agents. One of the
advantages of natural products is their low toxicity compared with
conventional chemo-drugs. Combination of natural compounds and
standard chemotherapeutic drugs may exert additive or synergistic
effects in killing cancer cells, which would in turn allow lower
and safer doses to be used. Here, we investigated a plant extract
for its activity against pancreatic cancer. Herbal preparation of
Pao Pereira (Pao), a rainforest tree in the family of Apocynaceae,
has long been used by oncologic patients and practitioners in
complementary and alternative medicine. However, its anticancer
activities against pancreatic cancer have yet to be systematically
studied. A β-carboline alkaloid-enriched extract of Pao has
recently been reported to suppress prostate cancer cells (10). Previous studies have shown the
potential of β-carboline alkaloids against several tumors (11–13).
In the present study, we investigated the activities of the
β-carboline alkaloid-enriched extract of Pao (14) against pancreatic ductal
adenocarcinoma, using multiple cell lines and a mouse model. The
combination effect of the extract with Gem was also
investigated.

Materials and methods

Cell lines and reagents

Human pancreatic cancer cell lines PANC-1, AsPC-1,
HPAF-II, BxPC-3 and MiaPaCa-2 were obtained from the American Type
Culture Collection (ATCC, Manassas, VA, USA). Immortalized human
lung epithelial cells MRC-5 were provided by Dr Sittampalam at the
University of Kansas Medical Center, and were used as a comparison
to cancer cells. All the cells were cultured at 37°C in 5%
CO2/95% air in recommended growth media containing 10%
fetal calf serum. Pao extract was provided by Natural Source
International (New York, NY, USA). Pao and Gem (Sigma, St. Louis,
MO, USA) were prepared in sterile water and stock at −20°C.

Cell viability assay

Cells were assessed for viability by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay at 72 h of treatment. Cells in exponential growth phase were
exposed to serial dilutions of Pao, Gem, or the combination of the
two, for 72 h. Then, cells were changed into fresh media containing
MTT and were incubated for 4 h. The colorimetric MTT assay assessed
relative proliferation, based on the ability of living, but not
dead cells, to reduce MTT to formazan (15,16).
Cells did not reach plateau phase during the incubation period.
Fifty percent inhibitory concentration (IC50) was
defined as the concentration of drug that inhibited cell growth by
50% relative to the untreated control. Pilot experiments for each
cell line were performed to optimize cell density and assay
duration and to center drug dilution series approximately on the
IC50.

Apoptosis detection by flow
cytometry

Cells were exposed to various concentrations of Pao
for 48 h. Cells were washed in PBS, resuspended in binding buffer,
and subjected to FITC-conjugated Annexin V and propidium iodide
(PI) staining according to the manufacturer’s protocol (BD
Biosciences, San Jose, CA, USA). Cells were analyzed by flow
cytometry. Annexin V positive cells and Annexin V-PI double
positive cells were identified as early and late stage apoptotic
cells, respectively. PI single-positive cells were identified as
necrotic cells.

Intraperitoneal pancreatic cancer mouse
model

Animal experiments were conducted following a
protocol (#2012–2035) approved by the Institutional Animal Care and
Use Committee. Through an intra-pancreas surgical procedure, the
human pancreatic ductal adenocarcinoma cells PANC-1 were
orthotopically implanted into the pancreas of nude mice
(3.2×105/mice). Ten days after tumor cells were
implanted, treatment began with intraperitoneal (i.p.) injection of
Gem (20 mg/kg, every 4 days), Pao (20 or 50 mg/kg daily), the
respective combination of Gem and Pao and saline as control. To
allow in vivo imaging, PANC-1 cells were transfected with
luciferase gene. Luciferin at 150 mg/kg was administered i.p. each
time prior to imaging for the luminance of tumor cells. Mice were
kept anesthetized with ~2.5% isoflurane. An IVIS in vivo
imaging system (Caliper Life Sciences, Hopkinton, MA, USA) was used
to scan the mice. The Living Image 4.1 software (Caliper Life
Sciences) was used for analysis of the images. After 70 days of
treatment, mice were euthanized. All tumor lesions in the
peritoneal cavity were collected and weighed. Major organs such as
liver, kidney and spleen were subjected to histological analysis
for any damage due to potential drug toxicity.

Data analysis

MTT data were normalized to their corresponding
untreated controls for each condition (drug and cell type) and were
expressed as percentage viability. Dose-reduction index (DRI)
values for Gem were calculated by the equation
DRIICx=(DGem/DGem+Pao), where
DGem is the dose of Gem alone required to produce an ICx
level of cytotoxicity, and the divisor DGem+Pao is the
dose of Gem needed to produce the same ICx level of cytotoxicity
when it is combined with Pao (at a given molar ratio).
DRIGem is defined with respect to Gem. Combination index
(CI) values were calculated by the equation CIICx =
(DGemCombo ICx/DGem ICx) +(DPaoCombo
ICx/DPao
ICx)+α[(DGemComboICx)(DPaoComboICx)/(DGem
ICx)(DPao ICx)], where D is the dose of Gem and
Pao either alone or in combination at a given constant ratio
required to produce an ICx level of cytotoxicity (17–19).
The more conservative assumption of mutual exclusivity was adopted
(α=0). SPSS15.0 was used for additional statistical analysis.

Results

Effect of Pao extract against pancreatic
cancer cells

Human pancreatic cancer cell lines (PANC-1, AsPC-1,
HPAF-II, MiaPaCa-2 and BxPC-3) were compared to an immortalized
epithelial non-tumorigenic cell line (MRC-5) for sensitivity to
Pao. All the cancer cells were susceptible to Pao treatment with
IC50 ranging from 75 to 215 μg/ml. MRC-5 cells were less
sensitive to the same treatment with an IC50 of 547
μg/ml (Fig. 1A). At the
concentration of 400 μg/ml Pao selectively killed 85–100% of cancer
cells, and only decreased viability in MRC-5 cells for 25%. Colony
formation in soft agar was used to assess the survival of
tumorigenic cancer cells, which has been positively correlated to
in vivo tumorigenicity of the cancer cells in animal models
(20,21). Untreated PANC-1 cells formed
colonies in soft agar at a rate of 12%. Pao at 400 μg/ml completely
inhibited formation of colonies of PANC-1 cells in soft agar
(Fig. 1B), indicating no survival
of tumorigenic cancer cells with this treatment.

To assess Pao-induced death pathway, Annexin V/PI
staining was performed to detect apoptosis vs. necrosis in PANC-1
cells treated with Pao. Flow cytometry demonstrated that the
percentage of cells positive with Annexin V/PI staining increased
from 4.3% in untreated cells to the highest, 96%, in 400 μg/ml
Pao-treated cells (Fig. 2A). The
induction of apoptosis was dependent on concentrations of Pao.
Apoptosis contributed to the majority of cell death, while necrosis
contributed to only 2–16.5% of cell death at all tested
concentrations. As a consistent finding, cleavage of caspase-8,
caspase-3 and PARP was also detected in Pao-treated PANC-1 cells.
The cleavage was induced dependent on the concentration of Pao and
time of treatment (Fig. 2B).

Synergistic effect of Pao in combination
with Gem against pancreatic cancer cells

After determining the dose-response relationships to
Pao in the cancer cells (Fig. 1A),
the dose-response relationships to Gem were also established in
PANC-1, AsPC-1, HPAF-II and BxPC-3 cells (Fig. 3A, dotted lines). A constant ratio
design was used to systematically examine the combination effect of
Gem and Pao (Gem+Pao). Ratio of Gem:Pao was chosen as
IC50Gem:IC50Pao. Cell viability was examined
after 72 h of treatment to obtain the optimum Gem effects. Results
clearly showed that when Pao was added to Gem, drops in cell
viability were markedly enhanced in all tested cells, compared to
Gem as a single agent (Fig. 3A).
The enhancement of the cytotoxic effect was particularly obvious in
AsPC-1 cells which were more resistant to Gem treatment.

The combination index (CI) for each cell type was
calculated based on the isobologram principle to examine whether
drug combinations may be synergistic (CI<1), additive (CI=1), or
antagonistic (CI>1) (17–19).
In addition, DRI for Gem was calculated to indicate whether Gem
concentration can be reduced when Pao was combined to produce the
same cytotoxic effect. DRI values of >1 indicate a favorable
combination. DRI<1 is interpreted as an antagonistic
combination. In all cell lines tested, CI values were ≤1 and DRIs
were >1 (Fig. 3B) across the
desired levels of effect (fraction affected, fa),
indicating additive to synergistic effect when Pao was combined
with Gem. These data also showed that the concentration of Gem is
decreased to produce an equitoxic effect on pancreatic cancer cells
when Pao is combined.

In vivo tumor inhibitory effect of Pao
either alone or in combination with Gem

An orthotopic pancreatic cancer mouse model was used
to evaluate the effect of Pao and Gem plus Pao (Gem+Pao) treatment.
Compared to a subcutaneous xenograft model, this model better
mimics clinical conditions of human pancreatic cancer, as the local
environment for pancreatic cancer development was represented.

Treatment was carried out as described in Materials
and methods. Tumor progress was monitored through longitudinal live
animal imaging. Representative images in 5 mice from each group are
shown in Fig. 4A. Gem at an early
stage of the treatment showed tumor inhibitory effects (day 20,
Fig. 4A), but it failed to inhibit
the tumor at a later stage (day 69, Fig. 4A). At the end of experiment,
Gem-treated mice had only insignificant reduction of tumor burden.
By contrast, Pao at either 50 mg/kg or a lower dose of 20 mg/kg
strongly inhibited tumor growth throughout the treatment period,
and resulted in significantly reduced tumor burden at the end of
experiment. Of note, the combination treatments mimicked the effect
of Pao treatment alone, without showing additional effects with
added Gem. These observations were confirmed by quantification of
bioluminescence intensity in all images to show total tumor burden
(Fig. 4B). Gem did not provide
significant inhibition of the tumor at the end of the treatment. By
contrast, a significant inhibition was shown between the control
group and the Pao-presenting treatment groups. There was no
difference whether Gem was present or absent in the Pao-treated
groups. Both low (20 mg/kg) and high dose (50 mg/kg) of Pao
provided similar effects. A dose-dependent effect to Pao was absent
at the tested doses.

At necropsy, all tumors were weighed and metastatic
lesions were counted. The results clearly confirmed data from live
animal imaging (Fig. 5). Whereas
Gem had minimal reduction on total tumor weight (P=0.25), Pao alone
decreased tumor weight by 72% at 20 mg/kg/day and by 70% at 50
mg/kg/day (P<0.05 for both groups vs. control) (Fig. 5A). The Pao-treated groups had
significantly lighter tumor burden compared to the Gem-treated
group (P<0.05). By combining Pao and Gem, tumor weight decreased
by 82% (Gem + 20 mg/kg Pao) and by 78% (Gem + 50 mg/kg Pao)
relative to control. Notably, the enhancement was not significant
compared to Pao single-drug treatment, but was highly significant
compared to Gem single-drug treatment. Again, there was no
dose-dependence to Pao, consistent with imaging.

Regarding metastasis, the effect mirrored what was
described above. Gem did not inhibit the formation of metastatic
lesions in mice. Pao at both tested doses markedly inhibited the
number of metastatic lesions (Fig.
5B). Pao treatment also decreased the number of mice that
developed metastasis disease (Fig.
5C), while Gem failed to do so. The combinations of Gem+Pao had
better effect compared to Gem in reducing either number of mice
having metastasis, or the number of metastatic lesions in the mice,
but had no difference compared to Pao treatment alone. There was no
difference between the different doses of Pao.

Proteins were isolated from tumor samples of the
treated and control mice. Western blot analysis showed cleavage of
caspase-3 and PARP in Pao and Gem+Pao treatment groups at all doses
(Fig. 5D). These results confirmed
the in vitro data that Pao induced apoptosis in tumor
cells.

Pao treatment possessed low toxicity as no toxic
effect was observed associated with the treatments. At the end of
the experiments, major organs (kidney, liver and spleen) were
subjected to haematoxylin and eosin (H&E) staining and
histological analysis. No tissue damage was detected in any of the
groups (Fig. 5E).

Discussion

Gemcitabine (Gem) as the first-line therapy of
pancreatic cancer provides little impact on the median survival for
patients with locally advanced or metastatic pancreatic cancer
(22,23). Our study consistently observed that
Gem only produced tumor inhibitory effects at early times of the
treatment course when the tumors were small, but it failed to
provide inhibition in tumor burden or metastasis in a longer time
in our animal model. By contrast, the extract of Pao Pereira (Pao)
exhibited strong inhibition in PANC-1 tumors throughout the course
of the experiment, reaching >70% inhibition even when tumors did
not respond to Gem anymore. Consistent with the in vitro
dose reduction effect for Gem, the combination of Pao and Gem had a
better effect than Gem in vivo. However, the combination did
not make a difference compared to Pao treatment alone in
vivo. This is likely due to the fact that tumors did not
respond to Gem treatment at a later course of the in vivo
experiment. The Gem non-responsive tumor still responded to Pao
treatment. These results greatly raise the potential for using Pao
in pancreatic cancer treatment, alone or in addition to Gem.

Cell death and apoptosis induction showed
dose-dependence on Pao concentrations in vitro, but such
dose-response was lacking in the in vivo tumor inhibitory
effects. The low (20 mg/kg) and high (50 mg/kg) doses of Pao had
similar tumor inhibition in mice bearing PANC-1 tumors. Mice
treated with 20 mg/kg Pao even had the tendency of fewer metastatic
lesions than mice treated with 50 mg/kg Pao. In another study using
Pao in prostate cancer, a similar dose-independence was observed
(10). The reason for this
treatment effect remains unknown. Since Pao was administered
through intraperitoneal injection in our study, this effect was not
likely due to the gastrointestinal absorption of bioactive
components. However, it is possible that high concentrations of Pao
could induce metabolic enzymes in vivo, potentially altering
the chemical profile of the bioactive components to an inactive
state. The effects of Pao in mice are probably a composite of
multiple organ systems, unlike cells in culture systems. It is also
possible that there is a threshold in the concentration that Pao
executes antitumor effects in vivo, and above the threshold
the effect might not be concentration-dependent, similar to the
situation of the concentration-independent antibiotics vancomycin
(24). Moreover, a U-shaped
dose-response relationship was proposed to exist between essential
nutrients and their biologic impact (25). Several studies have reported that a
non-linear relationship also applies to anticancer agents (26–29),
although the mechanisms/reasons causing this effect remain unclear.
Our data using the Pao extract appear to validate this
hypothesis.

In addition to the substantial inhibitory effect
against pancreatic cancer cells, Pao as a natural product had
relatively low toxicity towards normal cells. Low toxicity was
evident in mice treated with Pao where major organ toxicities were
absent. In addition, by the dose-reduction effect, Pao allowed for
lower concentrations of Gem while achieving an equivalent
cytotoxicity in cancer cells with higher Gem concentrations alone.
This may decrease the toxicity associated with chemotherapy.

Although the potential benefits of Pao were
suggested in our study, the mechanism(s) underlying the Pao-induced
anticancer effects warrant further study. A previous study reported
DNA damage and cell cycle inhibition by Pao in prostate cancer
cells (10). Our study showed that
Pao mainly induced apoptosis both in vitro and in
vivo as shown by flow cytometry and the cleavage of caspase-8,
caspase-3 and PARP, which can be inter-connected to DNA damage and
cell cycle inhibition. However, as this plant preparation contains
a complex mixture of natural compounds, there is potential that it
impacts multiple molecular targets and pathways that lead to
apoptosis.

Preponderance of bioactive compounds is often found
in medicinal plant and herbal mixtures, making them a notable
source for discovery of novel drug leads. The β-carboline-enriched
Pao extract could contain compounds that possess potent anticancer
activity. Data presented here is the initial step in identifying
the anticancer activity of Pao. Active components could be isolated
and developed for optimizing efficacy, toxicity and other profiles
that could lead to anticancer drug development.

Collectively, our data demonstrate the
anti-pancreatic cancer activity of the Pao Pereira extract in
vitro and in vivo. Further investigation is required for
the use of this plant extract in the treatment of pancreatic
cancer.

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